Complex evaluation of serum immunoglobulin levels in patients with chronic lymphocytic leukemia: Significant increase in IgA after first‐line chemoimmunotherapy

Abstract Introduction The impact of chemoimmunotherapy (CIT) on immunoglobulin (Ig) quantities in patients with chronic lymphocytic leukemia (CLL) has not been extensively studied. Methods We analyzed Ig levels in 45 stable patients with indolent CLL (without indication for treatment) and 87 patients with progressive disease before first‐line treatment. Fifty‐five patients were evaluated again after the treatment with CIT. Results We observed significantly lower levels of all Ig classes and subclasses in patients with progressive disease compared to patients with indolent disease. After treatment, median IgA increased from 0.59 g/L to 0.74 g/L (p = 0.0031). In stable patients, lower IgA2 was associated with shorter time to first treatment, although it did not reach statistical significance (p = 0.056). Shorter overall survival was observed in patients with progressive disease and lower IgG2 (p = 0.043). Surprisingly, among the patients with progressive CLL, unmutated IGHV genes were associated with higher levels of IgG, IgG1 and IgM, while TP53 mutation and/or 17p deletion were associated with higher levels of IgA and IgA1. Conclusions CIT may lead to increase in IgA levels. Hypogammaglobulinemia is more common in patients with progressive CLL and unmutated IGHV or TP53 dysfunction.

many prognostic markers, such as Rai or Binet stage, cytogenetic aberrations, mutational status of the immunoglobulin heavy chain variable region (IGHV), or mutation of the tumor protein 53 (TP53) gene, have been established to help identify patients with an early need for treatment. 2Another typical feature of CLL is a complex alteration of the immune system, resulting in progression of the disease, autoimmune complications, second malignancies, and a higher frequency of infections. 3,4Infections are the most important cause of morbidity and mortality in CLL patients, affecting more than 80% throughout the disease course, and are the cause of death in up to 60%. 4 The longest known immune defect in CLL patients is hypogammaglobulinemia, described since the 1960s. 5][8][9][10][11][12][13][14][15][16][17][18][19][20] Typical pathogens associated with hypogammaglobulinemia are encapsulated bacteria commonly causing respiratory infections: Staphylococcus aureus, Haemophilus influenzae, or Streptococcus pneumoniae. 21nly some researchers have investigated the possible association between hypogammaglobulinemia and prognostic factors.Neither age, sex, β2 microglobulin level, CD38 or ZAP70 positivity, nor unfavorable cytogenetic aberrations or unmutated IGHV were associated with low immunoglobulin (Ig) levels in a study by Mauro et al. 6 Others observed an association of hypogammaglobulinemia with advanced stage, CD49d positivity, or higher leukocyte count. 7,8he mechanism leading to low Ig levels in CLL patients is complex.CLL cells can inhibit bone marrow plasma cells through the CD95 (Fas receptor on plasma cells)-CD95 L (Fas ligand on CLL cells) interaction. 22dditionally, inhibition of plasma cells by autologous NK cells has been described. 23In a study of Criado et al., a direct correlation was found between total Ig levels and the number of normal residual B cells among CLL patients. 24Various defects in T cells and the related inability to form specific immune responses probably contribute as well. 25he last decade has seen chemoimmunotherapy (CIT) being replaced with targeted treatment with the Bcl-2 inhibitor venetoclax and Bruton tyrosine kinase inhibitors (BTKi).Nevertheless, some CIT regimens are still relatively widely used, especially where newer treatment options are less affordable. 2636][37][38] 1. 1 | Aims of the study To assess differences in Ig levels between patients with indolent disease (without indication for treatment) and patients with progressive disease before first-line treatment.In addition, changes after first-line treatment with CIT were evaluated, as well as associations between the Ig levels, disease course, known prognostic factors, and frequency of infections.

| Patients and data acquisition
All patients were diagnosed with CLL according to the 2008 International Workshop on CLL (IWCLL) criteria. 39All of them also signed informed consent forms, the study was approved by the local ethics committee and conducted according to the principles of the Declaration of Helsinki.The study had single-center design-all patients were prospectively followed at the 4th Department of Internal Medicine -Hematology, University Hospital in Hradec Králové, Czech Republic, from September 2013 to November 2020.
Samples of peripheral blood were collected from patients immediately before the treatment and 2-3 months after treatment completion.As for the patients with indolent disease course who were not treated, their samples were collected just upon entry into the study.None of the patients received any form of Ig replacement.The treatment indication was based on the IWCLL criteria. 39CIT regimens used were as follows: fludarabine + cyclophosphamide + rituximab (FCR), bendamustine + rituximab (BR), obinutuzumab + chlorambucil (O-Clb) and rituximab + chlorambucil (R-Clb).1][42] Information on known prognostic factors (Rai stage, fluorescence in situ hybridization (FISH) and karyotyping results, IGHV and TP53 mutational status), as well as information on frequency, severity, and site of infections were collected from patients' records.TP53 status was investigated by Sanger sequencing, IGHV mutational status was obtained after amplification of IGHV gene transcript by polymerase chain reaction with subsequent sequencing of product with Sanger method and comparing the result to germline genes in Ig databases.Time to first treatment (TTFT), time to next treatment (TTNT), and overall survival (OS) were calculated from the time of sample collection.Control group was established from healthy blood donors.
Serum levels of IgG, IgG subclasses IgG1-4, IgA, IgA subclasses IgA1-2, and IgM were measured by immunonephelometry using the immunochemistry system Image by Beckman Coulter (Miami, FL, USA).All patients also underwent serum protein electrophoresis, and if a narrow spike in the gamma zone was detected, immunofixation electrophoresis was added to detect a potential paraprotein.Measurements of patients with paraproteins were not included in the statistical analysis for a particular Ig class.

| Statistics
Different cohorts of patients were compared by use of the Mann-Whitney U-test, while paired samples of the same patients before and after the treatment were compared using the Wilcoxon test.Correlation analysis was used to assess relationship between Ig levels and age or absolute lymphocyte count (ALC).Association between Ig levels and OS, TTFT, or TTNT was explored with the Cox proportional hazards regression method.The Kaplan-Meier method was used for survival analysis.The ideal cut-off values for Ig quantities to allow for the best separation of the survival curves were determined by receiver operating characteristic (ROC) curve.Statistical significance was defined as p value ≤0.05 Statistical analysis was performed with MedCalc software, version 20 (MedCalc Software Ltd, Mariakerke, Belgium).

| RESULTS
Patients were enrolled into the study from September 2013 to May 2019, and then followed up until November 2020.Out of 126 enrolled patients, 45 were without indication for treatment (group with indolent, stable disease), while 81 had progressive CLL indicated for treatment.Six patients were analyzed as part of the stable cohort and later again, once their disease progressed.These six were not used for comparison between stable and progressive cohort, but they were included in comparison of Ig values before and after CIT.Blood sample after therapy was taken in 55 patients.The rest of them were treated with regimens other than R-Clb, O-Clb, BR or FCR (usually high-dose corticosteroids or targeted treatment) or their blood samples were lost or not taken for various reasons.The control group consisted of 45 healthy individuals (median age 61 years [range, 52-73], 44% males).Basic characteristics of all other cohorts are shown in Table 1 and flow chart describing the patients' disposition is provided in Figure 1.Partial or complete remission was achieved in 8/11 patients after R-Clb, 8/9 after O-Clb, 15/17 after BR and in all 18 patients after FCR.

| Comparison among controls, patients with stable and progressive disease and the effect of treatment
In the stable patient cohort (n = 45), one patient had IgG paraprotein.In patients with progressive disease (n = 87), paraproteins in the IgG/IgA/IgM class were detected in 7/1/5 patients.In patients analyzed before and after treatment (n = 55), paraproteins in the IgG/IgA/IgM class were detected in 3/0/3 patients.All patients with detected paraproteins were excluded from further analysis for a particular Ig class and its subclasses.All three patients' cohorts (stable, progressive and patients after treatment) had significantly lower levels of all Ig classes and subclasses than controls, with single exception of IgG3 (detailed in Tables S1-S3).The number of patients with hypogammaglobulinemia in each class and subclass is described in Table 2.
With the exception of IgG3, we observed significantly lower levels of all Ig classes and subclasses in patients with progressive disease (n = 81) compared to patients with stable disease (n = 45) (Table 3 and Figure 2).Regarding paired samples before versus after therapy (n = 55), the only significant change was an increase in IgA (Table 4 and Figure 2).As Ig levels did not change significantly after treatment (except for IgA), they remained significantly lower than those of stable patients (Table 5).

| Relation to infections and prognostic significance
In the 3-year period prior to blood collection, infection of any grade occurred in 22/45 stable patients.Sites of infection are described in Table 6.Ig levels of patients with and without infections were not significantly different (data not shown).
Out of 87 patient with progressive disease, 29 had experienced infections within the 3-year period prior to blood collection.As nine patients had multiple infections, there were a total of 48 episodes of infections (see Table 6 for details).Again, differences between Ig levels of patients with and without infections did not reach statistical significance.
As grade 3 or higher infections occurred only in four patients with stable disease and six patients with progressive disease, they were not analyzed separately.
Progression to treatment occurred in 12/45 stable patients and three of them died.The TTFT at 3 years was 79%.None of the Ig levels were significantly associated with TTFT, although there was a borderline p value in the case of IgA2 (p = 0.056).
Within the cohort of progressive CLL, 18/87 patients died and three-years OS was 78%.Shorter overall survival was observed in patients with lower IgG2 (p = 0.043).We performed ROC analysis to separate stable patients based on IgA2 level and patients with progressive disease based on IgG2 level into two groups with different TTFT and OS (Figure 3).We did not observe any association between Ig levels and TTNT or OS in 55 patients treated with CIT.Prognostic significance of increase in Ig quantity after treatment was also investigated.The increase was expressed as fold-change caused by treatment.In this analysis, higher increase of IgG2 was associated with shorter OS (p = 0.033).Other classes or subclasses of Ig had no influence on neither OS nor TTNT of treated patients.

| Association with prognostic markers
For patients with stable disease, the Ig levels of those with mutated and unmutated IGHV did not differ significantly.Other unfavorable prognostic factors (17p deletion, TP53 mutation and advanced Rai stage) were rarely present among stable patients -only two of them had advanced Rai stage, and none had 17p deletion or TP53 mutation.ALC inversely correlated with levels of IgA (p = 0.014) and IgM (p = 0.0024).There was no association between Ig levels and sex or age of stable patients.F I G U R E 2 Box and whisker plots comparing immunoglobulin levels in patients with stable and progressive disease (A-I) and IgA of patients before and after treatment (J).Quantities of immunoglobulins are expressed as medians in g/L.The boxes represent the interquartile range, and the whiskers are drawn at a distance of 1.5 times the interquartile range from the first and third quartiles.
When fold-changes after treatment were compared among groups of treated patients divided based on prognostic factors, only significant result was higher increase of IgA2 in patients with advanced disease (0.86-fold decrease in patients with Rai stage 0-II vs. 1.67-fold increase in patients with Rai stage III-IV, p = 0.0071).

| DISCUSSION
CLL and its treatment are associated with complex alteration of the immune system with a possible impact on the disease course, the risk of infections and thus the patients' prognosis.We present an analysis of Ig quantities, including subclasses, in patients with CLL before and after the first-line treatment with CIT.
In different studies, the prevalence of hypogammaglobulinemia in untreated patients was described,

T A B L E 6
Frequency of different infectious episodes.

Stable patients
Upper respiratory tract infection and bronchitis 12

Herpes labialis 3
Urinary  [6][7][8][9][10][11][12][13][14] Our results in patients with stable disease fit into this range, while in patients with progressive CLL, we found a higher prevalence of hypogammaglobulinemia for IgG (55%) and IgA (63%).Most of the published data are on patients at the time of diagnosis, which could explain the difference from our progressive cohort.In studies by Mauro et al. and Parikh et al., the prevalence of IgG deficiency increased during follow-up. 6,7We did not repeat measurements during follow-up (except for measurement right after treatment) but compared these with stable CLL with those indicated for treatment.The latter had significantly lower quantities of all Ig classes and subclasses.This might not be surprising, but as far as we know, a similar comparison has not yet been published.Lower IgG has been associated with shorter OS in studies by Rozman, Andersen and Crassini. 9,10,156,17 More often, an association between shorter OS and low levels of Ig was found for the IgA class. 9-12However, even this was not confirmed in all studies. 6,8,1711][12]15,17 Some studies evaluated the association of low Ig with shorter treatment-free survival or TTFT.1][12] In the publications of Singh et al. and Francis et al., no such association was found. 8,17In our cohort, we observed a trend towards shorter TTFT in stable patients with lower IgA2 and an association between low IgG2 and shorter OS in patients with progressive disease (Figure 3).Treated patients with higher relative increase in IgG2 also had shorter OS, which probably only reflects their lower pretreatment levels of this subclass.None of the main Ig classes had any prognostic value in our study, but it has to be said our number of patients was much lower than that in some abovementioned publications.Based on these studies, the association between Ig levels and prognosis remains controversial, but most evidence highlights the significance of IgA values.
Many of the aforementioned studies investigated the possible association of low Ig and infections, and the results were similarly ambiguous.Some studies did not find such an association in untreated patients with hypogammaglobulinemia. 10,13,18However, Andersen et al., with thus far probably the largest investigated cohort of 1204 untreated patients, observed a higher risk of infections in patients with low IgG, IgA, and IgM. 14Low IgA remained a significant risk factor even in multivariate analysis.In a study by Visentin et al., a higher frequency of major infections (requiring intravenous antibiotics or hospitalization) was associated with a combined deficiency of IgG with IgA or IgM. 19Additionally, Francis et al. and Freeman et al. observed more infections in patients with hypogammaglobulinemia, although some of them were pretreated, unlike in other works. 17,20In a cohort of 899 patients in Binet A stage CLL, the levels of Ig at the time of diagnosis had no association with infection; however, infections were more common in patients who developed hypogammaglobulinemia during follow-up (only IgG was assessed in this way). 6Finally, Ishdorj et al., who assessed a need for Ig replacement therapy rather than the frequency of infections, identified low IgG and IgA as independent predictors for future Ig replacement. 12We did not find any association between Ig levels and the frequency of infections, but again, the number of patients in our analysis was quite low, and more studies are necessary to definitively resolve this matter.The connection between Ig deficiency and infections might be difficult to establish because hypogammaglobulinemia is certainly not their only cause, and the inability of B cells to form a specific response to new antigens may also play a role. 43This is in line with the known lack of efficacy of vaccination in CLL. 44,45Naturally, numerous other changes in the immune system that occur during the disease course contribute to infections as well. 25e evaluated the possible association of Ig levels and the most widely used prognostic factors-Rai stage, IGHV mutational status, TP53 mutation, and deletion of 17p.Similar analyzes are quite rare in the literature.Parikh et al. observed lower IgG in patients in Rai stage III-IV, with higher CD49d expression and trisomy 12. 7 Other cytogenetic aberrations, age, sex, ZAP70 and CD38 expression or IGHV mutational status had no association with IgG levels.Neither of these factors, including Rai stage and β2microglobulin, were associated with IgG levels in a study by Mauro et al., but low IgA was more frequently found in patients with splenomegaly and CD38 positivity and low IgM was found in those with higher lymphocyte counts and in Rai stage 0-II. 6Ishdorj et al. found IgG and IgA deficiency more often in advanced Rai stage and in patients with IGHV 1-69, 3-21, and 3-49 subtypes. 12Low levels of IgA were also associated with CD38 positivity and elevated β2microglobulin, while abnormal IgM was associated with CD38 positivity and mutated IGHV.Cytogenetics were not assessed in this study.Finally, Singh et al. observed IgA deficiency in patients with advanced Rai stage and higher leukocyte count. 8Again, chromosomal aberrations were not assessed.
In our study, patients with CLL stage 0-II had levels of Ig comparable to those with stage III-IV.Here, we differ from the findings of Parikh et al., Ishdorj et al. and Singh  et al., but the explanation is simple.We divided patients into a stable cohort and a cohort with progressive disease.In the former, all but two patients were in the early disease stage, and this analysis was not performed.In the latter, no difference was found, but even patients with Rai stage I or II had some indication for treatment (e.g., massive lymphadenopathy) and therefore their disease activity was probably similar to that in patients with Rai stage III-IV.Indeed, when we evaluated all patients together, we observed lower levels of all Ig classes and subclasses, with the exception of IgG1 and IgG3, in patients with Rai stages III-IV (data not shown).Comparison of patients with stable and progressive disease proved to be a better discriminating factor, as all Ig subclasses, including IgG1 and IgG3, were significantly different between the two groups.
A rather surprising finding from our analysis is that out of patients with progressive disease, those with unfavorable prognostic factors had higher levels of some classes or subclasses of Ig.Patients with unmutated IGHV had higher levels of IgG, IgG1 and IgM, and those with TP53 mutation or 17p deletion had higher levels of IgA and IgA1.One possible explanation is that hypogammaglobulinemia is not directly connected to aggressiveness of the disease but rather to its duration.CLL with unfavorable biological features usually progresses more rapidly, and some changes to the immune system, including those resulting in hypogammaglobulinemia, might actually need more time to fully develop.To the best of our knowledge, our study is the only one that examined the possible influence of TP53 dysfunction.These results warrant further research on larger patient groups.
The most remarkable finding in our study is an increase in IgA levels after CIT administration.For the whole cohort of CIT-treated patients, median IgA increased from 0.59 g/L to 0.74 g/L (p = 0.0031), while other Ig levels did not change significantly.When we assessed treatment regimens separately, only in the FCR group did we observe an increase in IgA, IgG2 and IgA1, while changes after BR and chlorambucil-based regimens did not reach statistical significance.This may indicate that IgA increase in a whole cohort is mostly because of FCR-treated patients.Nevertheless, the numbers of patients in subgroups with different regimens were very small.Higher relative increase of IgA2 after treatment in patients with Rai stage III-IV compared to patients with stage 0-II (where there was actually decrease) is surprising, as base values before treatment between the groups were similar.The reason is not clear; however, as it was observed only in one minor subclass out of all the Ig subclasses, the importance of this finding is probably limited.
5][36][37][38] In some of these studies, patients with an increase in IgA also had fewer infections. 36,38In a randomized trial of ibrutinib versus ibrutinib plus rituximab, an increase in IgA was observed in both arms. 46Interestingly, IgG decreased in both arms, and IgM decreased in the combination arm (in other cited studies, IgG and IgM remained unchanged after ibrutinib).In contrast to BTKi, most studies report a decrease in Ig levels after CIT.Data from the era before the introduction of anti-CD20 treatment are fairly limited.In an analysis that included three different studies using fludarabine (n = 71) or fludarabine with prednisone (n = 103) as first-line treatment of CLL, the majority of patients had significant increases in their IgG and IgM levels. 27IgA levels mostly remained stable, although an increase was still more common than a decrease.These changes were independent of treatment response.More recent reports mostly encompass data from studies adding anti-CD20 treatment to chemotherapy.Keating et al. monitored Ig levels in their trial of FCR for treatment-naïve patients with CLL. 28They had no statistically significant change in IgG, IgA or IgM levels 6 months after treatment initiation.At 12 months, only IgG significantly decreased compared to the level before treatment (all three measurements of IgG level were performed in 105 patients).There was no association with treatment response.As the number of reports on Ig levels after CLL treatment is limited, we also included data from studies with other diagnoses.In a retrospective analysis of 211 patients with lymphomas (only 38 were CLL/small lymphocytic lymphoma) treated with a combination of rituximab and chemotherapy, the percentage of patients with low IgG increased from 15% to 48% after treatment. 29The proportion of patients with low IgA increased from 11% to 33% and that of patients with low IgM increased from 24% to 52%.A higher incidence of hypogammaglobulinemia was associated with a higher dose of rituximab and with fludarabine-containing chemotherapy but not with age, sex, or lymphoma type.In a small series of patients with indolent lymphomas treated with fludarabine and rituximab, 24 of 27 patients had no significant change in their IgG, IgA, or IgM levels. 30A large analysis of the effect of rituximab on IgG levels was performed on 243 patients with autoimmune conditions, mostly vasculitis. 31More than half of them also received oral cyclophosphamide.Before rituximab administration, 26% of patients had IgG <7 g/L, which increased to 56% during follow-up.There was no association between the total dose of rituximab and IgG nadir.The total dose of cyclophosphamide was associated with the IgG nadir in period after rituximab administration but not before it. 31ata on bendamustine or obinutuzumab treatment are scarce.In the GALLIUM study, patients with follicular lymphoma had a decrease in all Ig classes regardless of the antibody used or chemotherapy (bendamustine or cyclophosphamide with doxorubicin, vincristine and prednisone), but exact values were not published. 32In the GAUDI study, patients with follicular lymphoma received the same chemotherapy regimens combined with obinutuzumab only. 33The median levels of all Ig classes decreased after the induction phase of the treatment but remained within the normal range and relatively steady during obinutuzumab maintenance.No information is given on possible variation between different chemotherapies used.Taken together, apart from the trial of Keating et al., we do not have any data on Ig levels in large cohorts of CLL patients treated with currently used CIT regimens.
A possible explanation for the IgA increase after CIT administration lies in the general reconstitution of immune functions after suppressing the CLL clone.Even after ibrutinib treatment, where it has been described repeatedly, this phenomenon is poorly understood.Suggested mechanisms involve increased availability of growth factors for nonclonal B cells and their resulting increase after CLL cell elimination, changes in the T cell repertoire, or a shift from a Th2 to Th1 immune response. 36,47,48Although the Th1 type of immune response is based on cytotoxic T cells rather than antibody production, this shift was proven to correlate with IgA increase in ibrutinib-treated patients. 48t is possible that some of these changes are not exclusive to ibrutinib and its mechanism of action but may be attributed to any therapy that leads to disease remission.Apparently, more research is still needed to clarify the mechanism of IgA increase after ibrutinib and, as our study shows, possibly also after CIT.

| CONCLUSIONS
We performed a complex assessment of serum Ig levels in CLL patients, including the changes after the first-line treatment with CIT.To the best of our knowledge, our study is the first to report a comparison of Ig levels between patients with stable, indolent disease and disease indicated for treatment.All Ig levels were significantly lower in the latter group.We present hitherto unpublished observations of higher Ig levels in patients with TP53 dysfunction, possibly as a consequence of the shorter duration of a disease with an aggressive course.Finally, we complement scarce data on the impact of CIT on Ig levels in CLL.It appears that an increase in IgA levels described after ibrutinib may not be an exclusive feature of BTKi treatment but may be induced by CIT as well.Such an increase could at least partially be a consequence of suppressed disease activity.An IgA increase seems even more important in light of all the earlier published data suggesting a possible prognostic impact of IgA levels and their association with infectious complications.The major limitations of our study include the relatively low number of patients and its unicentric design.Thus, further studies in larger patient cohorts are warranted.

F I G U R E 1
Flow chart describing the patients' disposition.T A B L E 2 Number of patients with hypogammaglobulinemia in each class and subclass.

F
I G U R E 3 (A) Differences between TTFT of stable patients based on IgA2 level.ROC analysis set a cut-off level for the best separation of curves as 0.23 g/L.In the log-rank test, patients with IgA2 ≤ 0.23 g/L had a shorter TTFT (p = 0.0002).(B) Differences between OS of patients with progressive disease based on IgG2 level.ROC analysis set a cut-off level for the best separation of curves as 2.4 g/L.In the logrank test, patients with IgG2 ≤ 2.4 g/L had shorter OS (p = 0.0064).

Immunoglobulin class (lower limit of normal) Number of patients with hypogammaglobulinemia within each cohort Stable Progressive Before the treatment After the treatment
Note: Quantities of immunoglobulins are expressed as medians in g/L.
Comparison of patients before and after treatment (paired samples).Comparison of patients after treatment with patients with stable disease.
T A B L E 4 T A B L E 5Note: Quantities of immunoglobulins are expressed as medians in g/L.Abbreviation: NS, not significant.